The relationship between biological activity and primary structure of troponin I from white skeletal muscle of the rabbit.

1. A series of defined peptides which span the complete sequence were produced from troponin I isolated from white skeletal muscle of the rabbit. 2. Two peptides, CF1 (residues 64-133) and CN4 (residues 96-117) inhibited the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition was potentiated by tropomyosin and the Mg2+-stimulated adenosine triphosphatase of desensitized actomyosin. This inhibition, unlike that of troponin I and peptides derived from it, was not potentiated by tropomyosin. 4. The most active inhibitor, peptide CN4, was 45-75% as effective as troponin I when compared on a molar basis. The inhibitory peptide, CN4, and also whole troponin I were shown by affinity chromatography to interact specifically with actin. 5. A strong interaction with troponin C was demonstrated with peptide CF2 (residues 1-47), from the N-terminal region of troponin I. Somewhat weaker interactions were shown with peptides CN5 (residues 1-21) and with the inhibitory peptide CN4. 6. The significance of these interactions for the mechanisms of action of troponin I is discussed.     

Biological activities of bovine cardiac-muscle troponin C C-terminal peptide (residues 84-161).

1. Bovine cardiac-muscle troponin C was digested at cysteine residues 35 and 84, and the C-terminal peptide (residues 84-161) was isolated. 2. The C-terminal peptide contains two Ca2+-binding sites. These sites bind Ca2+ with a binding constant of 2.0 X 10(8) M-1. In the presence of 2 mM-Mg2+ the binding constant for Ca2+ is decreased to 3.7 X 10(7) M-1. The corresponding constants for native troponin C are 5.9 X 10(7) M-1. and 2.9 X 10(7) M-1 respectively. 3. Electrophoretic mobility of the C-terminal peptide is increased in the presence of 0.1 mM-CaCl2 as compared with the mobility in the presence of 2mM-EDTA. The same phenomenon was observed when electrophoresis was performed in the presence of 6 M-urea or 0.1% sodium dodecyl sulphate. 4. When saturated with Ca2+, the C-terminal peptide forms complexes with bovine cardiac-muscle troponin I both in the absence and in the presence of 6 M-urea. This complex is dissociated on removal of Ca2+. 5. The data suggest that the C-terminal peptide of troponin C contains two Ca2+/Mg2+-binding sites and interacts with troponin I. Thus, despite the 30% difference in amino acid composition, the properties of bovine cardiac-muscle troponin C C-terminal peptide are similar to those of rabbit skeletal-muscle troponin C C-terminal peptide.     

Structural and biological studies on synthetic peptide analogues of a low-affinity calcium-binding site of skeletal troponin C

We have synthesized four oligopeptides that are structural analogues of a low-affinity Ca2+-specific binding site (site II) of rabbit skeletal troponin C. One analogue (peptide 3) was a dodecapeptide with a sequence corresponding to the 12-residue Ca2+-binding loop (residues 63-74 in troponin C), two (peptides 4 and 5) were 23-residue in length, corresponding to residues 52-74 of the protein, and the fourth (peptide 6) was a 25-residue peptide corresponding to residues 50-74. All four peptides had one amino acid substitution within the 12-residue binding loop in which phenylalanine at position 10 was replaced by tyrosine to provide a marker for spectroscopic studies. In addition, peptides 3 and 4 each had a second substitution within the binding loop where glycine at position 6 was replaced by alanine. The second substitution was motivated by the conservation of glycine at the position in the Ca2+-binding loops of all four Ca2+-binding sites in troponin C. The peptides were characterized by their intrinsic fluorescence, ability to enhance the emission of bound Tb3+, affinity for Ca2+ and Tb3+, and circular dichroism. The affinity for Ca2+ was in the range 10-10(2) M-1, and the affinity for Tb3+ was in the range 10(4)-10(5) M-1. The binding constants of the longer peptides were several-fold larger than that of the dodecapeptide. With peptides 4 and 5, substitution of glycine by alanine at position 6 within the 12-residue loop decreased the affinity for Ca2+ by a factor of four, but had little effect on the affinity for Tb3+. However, the mean residue ellipticity of peptide 4 was substantially higher than that of peptide 5. Since peptide 4 differs from peptide 5 only in the substitution of glycine at position 6 in the loop segment, the conservation of glycine at that position may serve a role in providing a suitable secondary structure of the binding sites for interaction with troponin I. Peptides 4 and 6, when present in a large excess, mimic troponin C in regulating fully reconstituted actomyosin ATPase by showing partial calcium sensitivity and activation of the ATPase. Since these peptides are the smallest peptides containing the Ca2+-binding loop of site II, their biological activity suggests that a Ca2+-dependent binding site of troponin C for troponin I could be as short as the segment comprising residues 52-62.     

Biologically important interactions between synthetic peptides of the N-terminal region of troponin I and troponin C.

The interaction between troponin I and troponin C plays a critical role in the regulation of muscle contraction. In this study the interaction between troponin C (TnC) and the N-terminal region of TnI was investigated by the synthesis of three TnI peptides (residues 1-40/Rp, 10-40, and 20-40). The regulatory peptide (Rp) on binding to TnC prevents the ability of TnC to release the inhibition of the acto-S1-tropomyosin ATPase activity caused by TnI or the TnI inhibitory peptide (Ip), residues 104-115. A stable complex between TnC and Rp in the presence of Ca2+ was demonstrated by polyacrylamide gel electrophoresis in the presence of 6 M urea. Rp was able to displace TnI from a preformed TnI.TnC complex. In the absence of Ca2+, Rp was unable to maintain a complex with TnC in benign conditions of polyacrylamide gel electrophoresis which demonstrates the Ca(2+)-dependent nature of this interaction. Size-exclusion chromatography demonstrated that the TnC.Rp complex consisted of a 1:1 complex. The results of these studies have shown that the N-terminal region of TnI (1-40) plays a critical role in modulating the Ca(2+)-sensitive release of TnI inhibition by TnC.     

Calcium-induced changes in the location and conformation of troponin in skeletal muscle thin filaments.

Troponin is the regulatory protein of striated muscle. Without Ca2+, the contraction of striated muscle is inhibited. Binding of Ca2+ to troponin activates contraction. The location of troponin on the thin filaments and its relation to the regulatory mechanism has been unknown, though the Ca2+-induced dislocation of tropomyosin has been studied. By binding troponin(C+I) to actin in an almost stoichiometric ratio and reconstituting actin-tropomyosin-troponin(C+I) filaments, we reconstructed the three-dimensional structure of actin-tropomyosin-troponin(C+I) with or without Ca2+ from electron cryomicrographs to about 2.5 or 3 nm resolution, respectively. Without Ca2+, the three-dimensional map reveals the extra-density region due to troponin(C+I), which extends perpendicularly to the helix axis and covers the N-terminal and C-terminal regions of actin. In the presence of Ca2+, the C-terminal region of actin became more exposed, and troponin(C+I) became V-shaped with one arm extending towards the pointed end of the actin filament. This structure can be considered to show the location of troponin(C+I) in at least one of the states of skeletal muscle thin filaments. These Ca2+-induced changes of troponin(C+I) provide a clue to the regulatory mechanism of contraction.     

Biological activities of the peptides obtained by digestion of troponin C and calmodulin with thrombin.

1. Troponin C and calmodulin were not digested by thrombin at a significant rate in the presence of Ca2+. 2. In the presence of EGTA, troponin C was digested by thrombin to yield three peptides, TH1 (residues 1--120), TH3 (residues 1--100) and TH2 (residues 121--159). 3. In the presence of EGTA calmodulin was digested by thrombin giving two peptides, TM1 (residues 1--106) and TM2 (residues 107--148). 4. The electrophoretic mobilities of peptides TH1 and TM1 were increased at pH 8.6 by Ca2+ both in the presence and absence of urea. The mobilities of peptides TH2 and TM2 were unaltered under these conditions. 5. Peptides TH1, TH2 and tM1 formed complexes with troponin I on polyacrylamide gels at pH 8.6 in the presence of Ca2+. 6. The phosphorylation of troponin I by cyclic AMP-dependent protein kinase was significantly inhibited by peptides TH1 and TH3 and to a lesser extent by peptide TM1. 7. The calmodulin peptide TM1 activated myosin light-chain kinase when present in large molar excess. Peptide TM2 did not activate the enzyme.     

Ca2+-dependent interaction of the inhibitory region of troponin I with acidic residues in the N-terminal domain of troponin C

Ca2+ regulation of vertebrate striated muscle contraction is initiated by conformational changes in the N-terminal, regulatory domain of the Ca2+-binding protein troponin C (TnC), altering the interaction of TnC with the other subunits of troponin complex, TnI and TnT. We have investigated the role of acidic amino acid residues in the N-terminal, regulatory domain of TnC in binding to the inhibitory region (residues 96-116) of TnI. We constructed three double mutants of TnC (E53A/E54A, E60A/E61A and E85A/D86A), in which pairs of acidic amino acid residues were replaced by neutral alanines, and measured their affinities for synthetic inhibitory peptides. These peptides had the same amino acid sequence as TnI segments 95-116, 95-119 or 95-124, except that the natural Phe-100 of TnI was replaced by a tryptophan residue. Significant Ca2+-dependent increases in the affinities of the two longer peptides, but not the shortest one, to TnC could be detected by changes in Trp fluorescence. In the presence of Ca2+, all the mutant TnCs showed about the same affinity as wild-type TnC for the inhibitory peptides. In the presence of Mg2+ and EGTA, the N-terminal, regulatory Ca2+-binding sites of TnC are unoccupied. Under these conditions, the affinity of TnC(E85A/D86A) for inhibitory peptides was about half that of wild-type TnC, while the other two mutants had about the same affinity. These results imply a Ca2+-dependent change in the interaction of TnC Glu-85 and/or Asp-86 with residues (117-124) on the C-terminal side of the inhibitory region of TnI. Since Glu-85 and/or Asp-86 of TnC have also been demonstrated to be involved in Ca2+-dependent regulation through interaction with TnT, this region of TnC must be critical for troponin function.     

Cross-linking of rabbit skeletal muscle troponin with the photoactive reagent 4-maleimidobenzophenone: identification of residues in troponin I that are close to cysteine-98 of troponin C

We have used the sulfhydryl-specific, heterobifunctional, photoactivatable cross-linker 4-maleimidobenzophenone (BPMal) to study the interaction of rabbit skeletal muscle troponin C (TnC) and troponin I (TnI). TnC was specifically labeled at Cys-98 by the maleimide moiety of BPMal, and a binary complex was formed with TnI in the presence of Ca2+. Upon photolysis, covalent cross-links were formed between TnC and TnI [Tao, T., Scheiner, C.J., & Lamkin, M. (1986) Biochemistry 25, 7633-7639]. The cross-linked heterodimer was digested with cyanogen bromide, pepsin, and chymotrypsin into progressively smaller cross-linked peptides, which were purified by HPLC and then characterized by amino acid analysis and sequencing. We obtained a fraction from the initial CNBr digest that contained the expected peptide CB9 (residues 84-135) of TnC, cross-linked mainly to CN4 (residues 96-116), the "inhibitory region" of TnI. The peptides CN1 and CN3 of TnI were also detected in this fraction, but their molar ratios (compared to CB9) were only about 0.15 each, compared to 0.60 for CN4. Sequence analyses of fractions obtained after peptic and chymotryptic digests of the cross-linked CNBr fraction confirmed that CB9 and CN4 were the major cross-linked species. Quantitative analysis of sequencer results indicated that the residues in TnI that appeared to be most highly cross-linked to Cys-98 of TnC were Arg-108 and Pro-110, and to a lesser extent Arg-103 and Lys-107. These findings are consistent with previous studies on interactions between TnI and TnC and provide, for the first time, direct information on the identities of proximate amino acids in the two proteins.     

Calmodulin and troponin C: affinity chromatographic study of divalent cation requirements for troponin I inhibitory peptide (residues 104-115), mastoparan and fluphenazine binding

The different conformations induced by the binding of Mg2+ or Ca2+ to troponin C (TnC) and calmodulin (CaM) results in the exposure of various interfaces with potential to bind target compounds. The interaction of TnC or CaM with three affinity columns with ligands of either the synthetic peptide of troponin I (TnI) inhibitory region (residues 104-115), mastoparan (a wasp venom peptide), or fluphenazine (a phenothiazine drug) were investigated in the presence of Mg2+ or Ca2+. TnC and CaM in the presence of either Ca2+ or Mg2+ bound to the TnI peptide 104-115. The cation specificity for this interaction firmly establishes that the TnI inhibitory region binds to the high affinity sites of TnC (most likely the N-terminal helix of site III) and presumably the homologous region of CaM. Mastoparan interacted strongly with both proteins in the presence of Ca2+ but, in the presence of Mg2+, did not bind to TnC and only bound weakly to CaM. Fluphenazine bound to TnC and CaM only in the presence of Ca2+. When the ligands interacted with either proteins there was an increase in cation affinity, such that TnC and CaM were eluted from the TnI peptide or mastoparan affinity column with 0.1 M EDTA compared with the 0.01 M EDTA required to elute the proteins from the fluphenazine column. The interaction of these ligands with their receptor sites on TnC and CaM require a specific and spatially correct alignment of hydrophobic and negatively charged residues on these proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

A laser Raman spectroscopic study of Ca2+ binding to troponin C.

Laser Raman spectroscopy has been used detect structural changes in troponin C induced by Ca2+ binding. Addition of Ca2+ - Mg2+ sites produces perturbations in the amide III region of the spectrum indicative of increased alpha-helical content, and in regions of the spectrum corresponding to carboxylate, thiol, and phenol side chains. However, Ca2+ binding to the low affinity Ca2+ - specific sites is not detected by laser Raman spectral changes.     

Characterization of a region of the primary sequence of troponin C involved in calcium ion-dependent interaction with troponin I.

1. The CNBr digest of troponin C from rabbit fast skeletal muscle was shown to possess many of the functional properties of the whole troponin C molecule. 2. A peptide corresponding to residues 83-134 was isolated, which forms a Ca(2+-dependent complex with troponin I and neutralizes the inhibition by troponin I of the Mg(2+-stimulated adenosine triphosphatase of desensitized actomyosin. 3. The peptide inhibits the phosphorylation of fast-skeletal-muscle, but not cardiac-muscle, troponin I, by 3' :5'-cyclic AMP-dependent protein kinase. In this property it was as effective as whole skeletal-muscle troponin C when compared on a molar basis. 4. Biological activity was also present in other fractions obtained from the CNBr digest. 5. By gel filtration and affinity chromatography of the whole CNBr digest of troponin C, two peptides, one of which was identified as representing residues 83-134, were shown to form Ca(2+-dependent complexes with troponin I. 6. The significance of these findings for the mechanism of interaction of troponin C and troponin I is discussed.     

4Ca2+.troponin C forms dimers in solution at neutral pH that dissociate upon binding various peptides: small-angle X-ray scattering studies of peptide-induced structural changes.

Small-angle X-ray scattering data have been measured for rabbit skeletal muscle troponin C and its complexes with the venom peptides melittin and mastoparan as well as synthetic peptides based on regions of the troponin I sequence implicated in troponin C binding. At the neutral pH used in this study (pH 6.8), troponin C shows a tendency to form dimers in the presence of 4 mol equiv of Ca2+, but is monomeric in solution when 2 or less mol equiv of Ca2+ is present. The 4Ca2+.troponin C dimers dissociate upon binding melittin, mastoparan, and peptides based on residues 96-115, 1-30, and 1-40 in the troponin I sequence. This result suggests that the peptide-binding sites overlap with the regions of contact between troponin C molecules forming a dimer. Like the structurally homologous calcium-binding protein calmodulin, troponin C shows conformational flexibility upon binding different peptides. Upon binding melittin, troponin C contracts in a similar manner to calmodulin when it binds peptides known to form amphiphilic helices (e.g., melittin, mastoparan, or MLCK-I). In contrast, mastoparan binding to troponin C does not result in a contracted structure. The scattering data indicate troponin C also remains in an extended structure upon binding the inhibitory peptides having the same sequence as residues 96-115 in troponin I.     

Structure and dynamics of the C-domain of human cardiac troponin C in complex with the inhibitory region of human cardiac troponin I

Cardiac troponin C is the Ca2+-dependent switch for heart muscle contraction. Troponin C is associated with various other proteins including troponin I and troponin T. The interaction between the subunits within the troponin complex is of critical importance in understanding contractility. Following a Ca2+ signal to begin contraction, the inhibitory region of troponin I comprising residues Thr128-Arg147 relocates from its binding surface on actin to troponin C, triggering movement of troponin-tropomyosin within the thin filament and thereby freeing actin-binding site(s) for interactions with the myosin ATPase of the thick filament to generate the power stroke. The structure of calcium-saturated cardiac troponin C (C-domain) in complex with the inhibitory region of troponin I was determined using multinuclear and multidimensional nuclear magnetic resonance spectroscopy. The structure of this complex reveals that the inhibitory region adopts a helical conformation spanning residues Leu134-Lys139, with a novel orientation between the E- and H-helices of troponin C, which is largely stabilized by electrostatic interactions. By using isotope labeling, we have studied the dynamics of the protein and peptide in the binary complex. The structure of this inhibited complex provides a framework for understanding into interactions within the troponin complex upon heart contraction.     

Chymotryptic subfragments of troponin T from rabbit skeletal muscle. Interaction with tropomyosin, troponin I and troponin C

The binding of the chymotryptic troponin T subfragments to tropomyosin, troponin I, and troponin C was semiquantitatively examined by using affinity chromatography, and also by co-sedimentation with F-actin and polyacrylamide gel electrophoresis in 14 mM Tris/90 mM glycine. Circular dichroism spectra of the subfragments were measured to confirm that the subfragments retained their conformational structures. Based on these results, the binding sites of tropomyosin, troponin I, and troponin C on the troponin T sequence were elucidated. Tropomyosin bound mainly to the region of troponin T1 (residues 1-158) with the same binding strength as to the original troponin T. The C-terminal region of troponin T (residues 243-259) was the second binding site to tropomyosin under physiological conditions. The binding site of troponin I was concluded to be the region including residues 223-227. The binding of troponin C was dependent on Ca2+ ion concentration. The C-terminal region of troponin T2 (residues 159-259) was indicated to be the Ca2+-independent troponin C-binding site and the N-terminal side of troponin T2 to be the Ca2+-dependent site.     

Various properties of cardiac troponin C tryptic peptides

Using chromatography and preparative polyacrylamide gel electrophoresis, tryptic peptides TP 1 (residues 47-83), TP 2 (residues 84-118) and TP 3 (residues 119-161) were isolated in a highly homogeneous state from cardiac troponin C. Peptides TP 1, TP 2 and TP 3 were found to contain isolated cation-binding sites II, III and IV of cardiac troponin C. The interaction of these peptides with troponins I and T was studied. It was found that only peptide TP 2 could interact with troponin I. Neither of the peptides isolated interacted with troponin T. The cation-binding properties and structural peculiarities of peptide TP 1 were investigated. It was shown that despite its small size (37 amino acid residues), peptide TP 1 retained its ability to bind Ca2+ which caused conformational changes in the peptide structure. This was accompanied by changes in the electrophoretic mobility and absorption of TP 1 on phenyl-Sepharose.     

Effect of calcium ions on the interaction of troponin C with rabbit skeletal muscle troponin I and troponin T immobilized on polyvinyl chloride

Using a new methodological approach based on the binding of 125I-labeled troponin C to troponins I and T immobilized on polyvinylchloride, the Ca2+-dependent interaction of troponin components was investigated. In the absence of Ca2+, two types of sites of troponin C--troponin T interaction were revealed (Kd = 3.6.10(-8) M and 5.10(-7) M). It was found that Ca2+ induced the formation of a troponin I--troponin C complex which was resistant to 5 M urea (Kd = 4.10(-8) M). In the absence of Ca2+, the binary troponin T--troponin C complex also revealed two types of interaction sites (Kd = 7.1.10(-8) M and 2.10(-7) M); however, in the presence of Ca2+ only high affinity sites whose number increased almost 2-fold were revealed. The events that may take place in the whole troponin complex during Ca2+ binding by troponin C are discussed.     

The biological importance of each amino acid residue of the troponin I inhibitory sequence 104-115 in the interaction with troponin C and tropomyosin-actin

To systematically evaluate the contribution of each amino acid residue of the troponin I (TnI) inhibitory region (104-115), 14 synthetic analogs were synthesized by the solid-phase method. The analogs consisted of either single glycine or multiglycine replacements. The importance of the substituted amino acid(s) was determined from the extent of inhibition of the acto-S1 ATPase activity and the strength of binding to a troponin C (TnC) high pressure liquid chromatography affinity column of each synthetic analog. Every residue of the TnI sequence (104-115) is necessary to achieve maximum inhibition of the ATPase activity. However, the analogs quantitatively differed in the amount of inhibition induced. The TnI analogs bound less tightly to the TnC affinity column than the native synthetic peptide indicating that all residues in the TnI sequence contribute to the binding of TnC in the presence of Mg2+ or Ca2+. In the presence of Ca2+, there is a definite increase in the strength of the interaction between most analogs and TnC. This is accompanied with a shift toward a more specific interaction with the C terminus of the TnI inhibitory sequence.     

Investigation of cation-binding properties of cardiac troponin C peptides by circular-dichroism spectroscopy

Bovine cardiac troponin C was cleaved at residues cysteine-35 and cysteine-84. Three peptides, N-terminal (residues 1-34), central (residues 35-83) and C-terminal (residues 84-161), of cardiac troponin C were obtained in a homogeneous state. Saturation of troponin C or its C-terminal peptide with Ca2+ or Mg2+ is accompanied by an increase in the ellipticity at 222 nm in the c.d. spectrum. The half-maximal changes in the ellipticity of troponin C were observed at 32 nM-Ca2+ or 56 microM-Mg2+. The corresponding values for the C-terminal peptide are 7.1 nM for Ca2+ and 4.5 microM for Mg2+. The ellipticity of the central peptide (residues 35-83) containing the second cation-binding site was decreased on saturation with Ca2+. The half-maximal changes in the ellipticity occur at 80 microM-Ca2+. Study of the c.d. spectra suggests that the alpha-helices flanking the second cation-binding site of cardiac troponin C exist independently of Ca2+. Saturation of the third and fourth sites with these cations is associated with a considerable increase in the alpha-helix content, probably due to the formation of an alpha-helix flanking the third site on the N-terminus.     

The primary structure of troponin T and the interaction with tropomyosin.

1. Eight peptides were separated from the CNBr digest of troponin T from rabbit white skeletal muscle and characterized. 2. By study of the amino acid sequence of the methionine-containing peptides isolated after chymotryptic and tryptic digestion and of the N- and C-terminals of the CNBr peptides, six of the latter were shown to be arranged in the sequence CNB1-CNB2-CNB5-CNB6-CNB8-CNB7. The other two peptides, CNB1' and CNB3, have been shown to be partial digestion products. 3. The CNBr peptides CNB1' and CNB2 contained a common sequence and were the only peptides in CNBr digests of troponin T that formed a complex with tropomyosin as judged by viscometric and electrophoretic studies. 4. It is concluded that tropomyosin interacts with the N-terminal half of the troponin T molecule approximately in the region lying between residues 70 and 160. 5. Electrophoretic evidence indicates that tropomyosin and troponin C interact with troponin T. 6. None of the major CNBr peptides of troponin T isolated formed a complex with troponin C on electrophoresis at pH 8.6.     

Localization of a trifluoperazine binding site on troponin C

Trifluoperazine (TFP) was shown to interact with the cyanogen bromide fragment 9 (CB9) (residues 84-135) of rabbit skeletal troponin C and with a synthetic peptide representing the N-terminal region of CB9. The phenothiazine did not affect the calcium binding property of CB9 as observed by proton magnetic resonance and circular dichroism spectroscopies. The calculated calcium binding constants for CB9 in the presence and absence of trifluoperazine were identical (KCa2+ = 1.3 X 10(5) M-1). Localization of the trifluoperazine binding site was achieved by analyzing the 1H NMR spectrum of CB9 and of a synthetic fragment corresponding to residues 90-104 of CB9. Drug-induced shifting and broadening of the ring protons of phenylalanine residues and the methyl resonances of alanine, leucine, and isoleucine residues suggest that the segment 95-102 is in close proximity to the phenothiazine aromatic region. The neighboring negative side chains in the peptide sequence also suggest that the single positive charge present on the piperazine nitrogens of trifluoperazine may interact with them and sterically block a region of interaction of calmodulin (CaM) and troponin C (TnC) with modulated proteins such as phosphodiesterase. Primary sequence analysis of CaM and troponin C reveals that a homologous hydrophobic region to site 3 is also found in the N-terminal region of site 1 of both calcium binding proteins. Binding of TFP to CB9 occurs both in the presence and absence of calcium since the hydrophobic region in these small fragments is completely accessible to TFP whether calcium is present or not. The dissociation constant of the drug to apoCB9 (8 microM) was obtained by ellipticity measurements at 222 nm and was comparable to the 5 microM value obtained by Levin and Weiss [Levin, R. M., & Weiss, B. (1978) Biochim. Biophys. Acta 540, 197-204] for calcium-saturated rabbit skeletal troponin C.